This proposal will qualitatively describe and quantify how the volatile anesthetics depress various Ca2+ currents, and how such changes may contribute to depression of excitation-secretion coupling, ultimately contributing to the anesthetic state. Initially, selective toxins will be used to block specific channels expressed in a variety of cells, so that anesthetic actions on the isolated remaining Ca channel class will be examined. The mechanism by which volatile anesthetics actually alter Ca channel behavior will be investigated by two methods. a) Single channel kinetics in the presence of anesthetics will be examined to provide insights into how the rate of the molecular rearrangements and modes is altered by the anesthetics. b) Specific Ca channel types as defined by the alpha1 subunit, which forms the pore of the channel, will be expressed in Xenopus oocytes with and without the full complement of subunits, for study of anesthetic actions in isolation. Since the other Ca channel subunits (beta and alpha2-delta) tend to modulate (typically increase) Ca2+ current amplitude, one possibility is that anesthetics may "uncouple" the subunit(s) from the alpha1 component to mediate the actions. To test the hypothesis that transmitter release is reduced by an anesthetic exclusively by its depression of Ca2+ currents, two primary techniques will be utilized. a) Isolated synaptosomes will be employed in which glutamate release as well as Ca2+ levels can be measured. Comparison of anesthetic actions on Ca2+ entry with the amount of activated glutamate release will permit determination of whether mere blockade of Ca2+ entry accounts for all of the effects or whether subsequent steps in stimulus- section coupling may have a role in anesthetic action. b) Release of norepinephrine by chromaffin cells will be determined by a polarized oxidizing electrode to correlate changes in release with changes in Ca2+ currents caused by anesthetics. Protein kinase C (PKC) activity has been found to modulate Ca channel activity in a variety of settings, as well as to increase inhibitory GABAA Cl- currents. Since anesthetics have been found to diminish PKC activity, PKC inhibition may be a major anesthetic mechanism. Anesthetic effects on Ca channels in the presence of PKC activation (with phorbol esters) and inhibition will be measured to determine how loss of this modulatory system alters anesthetic effects. To correlate further how PKC may contribute to the anesthetic state, anesthetic-mediated effects on GABAA receptors, a known site of anesthetic action, will also be explored under different levels of PKC activation. Since narcotics and alpha2-adrenoceptors also depress Ca currents, the interaction of these analgesic drugs with volatile anesthetics, commonly employed together in clinical anesthesia, will be determined. The combined action of opiate or alpha2-adrenoceptors analgesics will be examined. The presence of additive, synergistic, or possibly canceling effects may provide insights into a better understanding of their combined use clinically.